The overall goal of this research is to determine the conformation of the M2 protein from the influenza A virus. Influenza A virus is a major public health concern, both in its annual death toll and its potential to cause devastating pandemics. The M2 protein is an essential element of the virus and serves as a proton channel. The channel is the target of the antiviral drugs amantadine and rimantadine, which block proton channel activity and inhibit virus replication. These antiviral drugs have proven effective for prophylaxis in prior influenza pandemics. The N-terminal extracellular domain of the M2 protein has striking sequence conservation across all human influenza A strains and is being developed as a target for a universal flu vaccine. A full understanding of the function of M2 and the biology of influenza relies on understanding the structure of this protein. Furthermore, as a relatively small membrane protein, the M2 protein is an experimentally tractable and valuable biophysical model for understanding ion channel function and as a testing ground for exploring the basic principles of membrane protein folding. [unreadable] [unreadable] Our strategy for studying the conformation of M2 involves synthetically incorporating spin labels at a series of sites within the protein, reconstituting the labeled protein into lipid membranes and measuring the electron paramagnetic resonance (EPR) spectral properties of the labels. Several types of structural information can be extracted from EPR studies: patterns of label mobilities across a protein sequence can be used to characterize topographic regions of a protein fold, accessibility of a label to collision with paramagnetic reagents of varying bilayer/aqueous solubility can also be used to determine the depth of a spin-labeled residue in the membrane, and the distance between two labels can be determined from the measurement of spin-spin couplings. [unreadable] [unreadable] The transmembrane domain, C-terminal cytoplasmic domain and the N-terminal extracellular domain of the M2 protein will each be examined in turn. EPR data will be collected in a variety of lipid bilayer compositions and in the presence of the antiviral drug amantadine. The M2 channel is pH-gated and the spin-labeled protein will be examined at several pH values to probe for conformational changes related to channel gating. [unreadable] [unreadable] [unreadable]